Methods for Analyzing Cysteamine Compositions

ABSTRACT

Methods of analyzing purity of compositions comprising cysteamine and detecting impurities in cysteamine compositions are described.

This application is a continuation of U.S. patent application Ser. No.14/306,820 filed Jun. 17, 2014, which claims the priority benefit ofU.S. Provisional Patent Application No. 61/835,987 filed Jun. 17, 2013,each of which is herein incorporated by reference.

FIELD OF THE INVENTION

The invention relates generally to methods for analyzing purity ofcompositions comprising cysteamine and detecting impurities incysteamine compositions.

BACKGROUND

Cysteamine (HS—CH₂—CH₂—NH₂) is able to cross cell membranes easily dueto its small size. At present, cysteamine is FDA-approved for thetreatment of cystinosis, an intra-lysosomal cystine storage disorder. Incystinosis, cysteamine acts by converting cystine to cysteine andcysteine-cysteamine mixed disulfide which are then both able to leavethe lysosome through the cysteine and lysine transporters respectively(Gahl et al., N Engl J Med 2002; 347(2):111-21). Treatment withcysteamine has been shown to result in lowering of intracellular cystinelevels in circulating leukocytes (Dohil et al., J. Pediatr 148(6):764-9,2006).

Impurities can be present in cysteamine formulations due to byproductformation during the manufacturing process and/or due to modification(e.g., degradation) during storage of the drug product. Accurate andcomplete determination of the purity of cysteamine-containingformulations is important in securing marketing approval for thepharmaceutical product and in demonstrating that the pharmaceuticalproduct has acceptable impurity levels for human administration at thetime of release and that it has acceptable storage stability. Analysisof the purity of cysteamine-containing formulations using an HPLC methodwith an electrochemical detection system has proven not to have thesensitivity required to detect certain impurities found in cysteamineformulations.

The present invention provides improved methods for analyzing purity ofcompositions comprising cysteamine and detecting impurities incysteamine compositions.

SUMMARY

The invention is directed to a method of analyzing purity ofcompositions comprising cysteamine. The method comprises injecting asample solution comprising cysteamine onto a reverse-phase HPLC column;eluting the sample from the column using a mobile phase comprising analkyl sulfonic acid (e.g., 1-hexanesulfonic acid and/or 1-octanesulfonicacid), a buffer, acetonitrile, and methanol; and measuring the elutedsample using a UV detector. The eluted sample is measured at awavelength of about 170 nm to about 250 nm.

In related methods, the invention provides a method of analyzing purityof delayed-release cysteamine formulations, such as enteric-coatedcysteamine beads. Such beads include beads loaded in individual-dosecapsules that are formulated for oral administration to a patient. Themethod comprises grinding enteric-coated cysteamine beads; dissolvingthe ground beads in a solvent having an acidic pH to form a samplesolution comprising cysteamine; injecting the sample solution onto areverse-phase HPLC column; eluting the sample from the column using amobile phase comprising an alkyl sulfonic acid (e.g., 1-hexanesulfonicacid and/or 1-octanesulfonic acid), a buffer, acetonitrile, andmethanol; and measuring the eluted sample using a UV detector at awavelength of about 170 nm to about 250 nm.

Additionally, the invention provides a method of analyzing purity ofcompositions comprising cysteamine comprising (i) dissolving acysteamine sample in a solvent having an acidic pH to form a samplesolution comprising cysteamine; (ii) injecting the sample solution ontoa C18 reverse-phase HPLC column; (iii) gradient eluting the sample fromthe column using first and second mobile phases, wherein the firstmobile phase comprises about 85% by volume of an aqueous solution havinga pH of about 2.6, the aqueous solution comprising about 23.6 mM1-octanesulfonic acid sodium and about 29 mM sodium phosphate; about 3%by volume of acetonitrile; and about 12% by volume of methanol, and thesecond mobile phase comprises about 10% by volume of an aqueous solutionhaving a pH of about 2.6, the aqueous solution comprising about 0.2 M1-octanesulfonic acid sodium and about 0.1 M sodium phosphate; about 18%by volume of acetonitrile; and about 72% by volume of methanol; and (iv)measuring the eluted sample using a UV detector at a wavelength of about210 nm or less.

Further aspects of the invention may become apparent to those skilled inthe art from a review of the following detailed description, taken inconjunction with the appended claims. While the invention is susceptibleof embodiments in various forms, described hereinafter are specificembodiments of the invention with the understanding that the disclosureis illustrative, and is not intended to limit the invention to specificembodiments described herein. The entire document is intended to berelated as a unified disclosure, and it should be understood that allcombinations of features described herein are contemplated, even if thecombination of features are not found together in the same sentence, orparagraph, or section of this document. For example, where embodimentsconcerning a method of analyzing purity of compositions comprisingcysteamine are described, embodiments involving methods of analyzingdelayed release cysteamine formulations such as enteric-coatedcysteamine, and the like that have the same properties and features arespecifically contemplated, and the reverse also is true.

In addition to the foregoing, the invention includes, as an additionalaspect, all embodiments of the invention narrower in scope in any waythan the variations specifically mentioned above. With respect toaspects of the invention described as a genus, all individual speciesare individually considered separate aspects of the invention. Withrespect to elements described as a selection within a range, it shouldbe understood that all discrete subunits within the range arecontemplated as an embodiment of the invention. Ranges may be expressedherein as from “about” or “approximately” one particular value and/or to“about” or “approximately” another particular value. When such a rangeis expressed, another embodiment according to the invention includesfrom the one particular value and/or to the other particular value.Similarly, when particular values are expressed as approximations, butuse of antecedents such as “about,” “at least about,” or “less thanabout,” it will be understood that the particular value forms anotherembodiment.

With respect to aspects of the invention described or claimed with “a”or “an,” it should be understood that these terms mean “one or more”unless context unambiguously requires a more restricted meaning. Theterm “or” should be understood to encompass items in the alternative ortogether, unless context unambiguously requires otherwise. If aspects ofthe invention are described as “comprising” a feature, embodiments alsoare contemplated “consisting of” or “consisting essentially of” thefeature.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides an HPLC chromatogram of cysteamine obtained according tothe methods described herein.

FIG. 2 provides HPLC chromatograms of cysteamine from Sample A obtainedusing a UV detector (A) and using an electrochemical detector (B).

FIG. 3 provides HPLC chromatograms of cysteamine from Sample B obtainedusing a UV detector (A) and using an electrochemical detector (B).

DETAILED DESCRIPTION

The invention provides improved methods for analyzing purity ofcompositions comprising cysteamine and detecting impurities incysteamine compositions. Such methods preferably provide highersensitivity and less selectivity relative to methods not in accordancewith the invention. By providing less selectivity, the inventive methodsallow detection of additional impurities than are detected by moreselective methods. Thus, the inventive methods can provide a morecomplete and more accurate determination of the number and amounts ofimpurities and/or related substances present in a cysteamineformulation. As used herein, the term “cysteamine formulation,”“cysteamine composition” or “composition comprising cysteamine” refersto any formulation comprising cysteamine, including formulationscomprising cysteamine salts. As used herein, the term “relatedsubstances” refers to compounds derived from cysteamine that are presentin the cysteamine formulation initially upon preparation and/or afterstorage. Cysteamine related substances include, but are not limited to,cysteamine dimer (i.e., cystamine) and other products obtained bymodification of cysteamine and/or reaction of cysteamine with othercomponents present in the formulation such as salts.

The invention is described in further detail below. Section headings arefor convenience of reading and not intended to be limiting per se.

HPLC Methods

In one aspect, the invention provides a method for analyzing purity ofcompositions comprising cysteamine and detecting impurities incysteamine compositions. The method comprises: injecting a samplesolution comprising cysteamine onto a reverse-phase HPLC column; elutingthe sample from the column using a mobile phase comprising an alkylsulfonic acid, a buffer, acetonitrile, and methanol; and measuring theeluted sample using a UV detector at a wavelength of about 170 nm toabout 250 nm. Optionally, the method includes dissolving a cysteaminesample in a solvent having an acidic pH to form the sample solutioncomprising cysteamine.

In a related aspect, the invention provides a method for analyzingpurity of enteric-coated cysteamine beads and detecting impurities inenteric-coated cysteamine beads. The method comprises: grindingenteric-coated cysteamine beads; dissolving the ground beads in asolvent having an acidic pH to form a sample solution comprisingcysteamine; injecting the sample solution onto a reverse-phase HPLCcolumn; eluting the sample from the column using a mobile phasecomprising 1-octanesulfonic acid, a buffer, acetonitrile, and methanol;and measuring the eluted sample using a UV detector.

In various embodiments, the solvent in which the cysteamine sample isdissolved comprises an alkyl sulfonic acid, a buffer, acetonitrile, andmethanol. Exemplary solvents include the HPLC mobile phase. When thesample is gradient eluted using first and second mobile phases, thesolvent is typically the mobile phase having the higher volumepercentage of aqueous components and the lower volume percentage oforganic components (generally referred to as the first mobile phase ormobile phase A).

The sample solution is injected onto a reverse-phase HPLC column.Typically, the sample solution is injected in a volume of 10 μL or 100μL, however, other injection volumes can be used (e.g., 5 μL, 20 μL, 30μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, or 90 μL of sample solution) andmultiple injections may be used (e.g., two, three, four, or fiveinjections of 10 μL of sample solution). A sufficient amount of sampleis injected onto the column such that substances related to cysteaminemay be detected. Generally, the amount of cysteamine injected onto thecolumn is at least 30 μg, at least 40 μg, at least 50 μg, and/or atleast 60 μg.

The HPLC column includes a packing material (i.e., a stationary phase).In reverse-phase HPLC, the column contains a hydrophobic packingmaterial. Exemplary reverse phase HPLC columns include, but are notlimited to, a C18 column, a C8 column, a silica column, a cyano-bondedsilica column, and a phenyl-bonded silica column. The HPLC columngenerally has a packing material particle size of about 1 μm to about 10μm in diameter, about 1 μm to about 7 μm in diameter, about 2 μm toabout 5 μm in diameter, about 3 μm to about 4 μm in diameter, and/orabout 3.5 μm in diameter. Typical HPLC columns have an internal diameterof about 0.1 mm to about 10 mm, about 0.1 mm to about 7 mm, about 0.15mm to about 5 mm, about 0.3 mm to about 5 mm, about 0.5 mm to about 5mm, about 0.7 mm to about 5 mm, about 1 mm to about 5 mm, about 1.5 mmto about 5 mm, about 2 mm to about 5 mm, about 3 mm to about 5 mm, about4 mm to about 5 mm, and/or about 4.6 mm. Generally, HPLC columns have alength of about 5 mm to about 500 mm, about 10 mm to about 250 mm, about20 mm to about 250 mm, about 30 mm to about 250 mm, about 50 mm to about250 mm, about 75 mm to about 200 mm, and/or about 100 mm to about 150mm.

Commercial HPLC columns include XBRIDGE analytical columns (Waters,Milford, Mass.), for example, XBRIDGE analytical columns having aninternal diameter and a length, respectively, of 1.0×50 mm, 1.0×100 mm,1.0×150 mm, 2.1×10 mm, 2.1×10 mm, 2.1×20 mm, 2.1×30 mm, 2.1×50 mm,2.1×75 mm, 2.1×100 mm, 2.1×150 mm, 3.0×20 mm, 3.0×30 mm, 3.0×50 mm,3.0×75 mm, 3.0×100 mm, 3.0×150 mm, 4.6×20 mm, 4.6×30 mm, 4.6×50 mm,4.6×75 mm, 4.6×100 mm, 4.6×100 mm, 4.6×150 mm, and 4.6×250 mm. XBRIDGEanalytical columns contain packing material having particle sizes, forexample, of about 2.5 μm in diameter, about 3.5 μm in diameter, andabout 5 μm in diameter.

The sample is eluted from the column using a mobile phase comprising analkyl sulfonic acid, a buffer, acetonitrile, and methanol. Typically,the elution is a gradient elution. During a gradient elution, first andsecond mobile phases having different compositions are used. The firstand second mobile phases are mixed in changing ratios throughout theelution such that the composition of the mobile phase changes over thecourse of the elution. A gradient elution optionally includes one ormore stepped changes in the ratio of the mobile phases.

An aqueous solution comprising an alkyl sulfonic acid and a buffertypically is included in the mobile phase in an amount of about 5% toabout 95% by volume of the mobile phase, for example, about 10% to about90% by volume, about 15% to about 85% by volume, about 20% to about 80%by volume, about 25% to about 75% by volume, about 30% to about 70% byvolume, about 35% to about 65% by volume, about 40% to about 60% byvolume, and/or about 45% to about 55% by volume. Other amounts of theaqueous solution may also be used. For example, when the sample isgradient eluted, one mobile phase may include the aqueous solution at alower concentration and the second mobile phase may include the aqueoussolution at a higher concentration. Thus, other exemplary amounts ofaqueous solution include about 75% to about 95% by volume, about 80% toabout 90% by volume, about 85% by volume, about 5% to about 30% byvolume, about 5% to about 25% by volume, about 5% to about 20% byvolume, and/or about 5% to about 10% by volume. The aqueous solutiontypically has an acidic pH, for example a pH of about 1.5 to about 6.5,about 2 to about 6, about 2 to about 5, about 2 to about 4, about 2 toabout 3, and/or about 2.4 to about 2.8.

Suitable alkyl sulfonic acids include, but are not limited to,ethanesulfonic acid, propanesulfonic acid, butanesulfonic acid,pentanesulfonic acid, hexanesulfonic acid, heptanesulfonic acid,octanesulfonic acid, nonanesulfonic acid, and decanesulfonic acid. Thealkyl sulfonic acid (e.g., 1-hexanesulfonic acid or 1-octanesulfonicacid) may be included in the aqueous solution of the mobile phase in anyof its salt forms such as in a sodium salt form, for example,1-hexanesulfonic acid sodium or 1-octanesulfonic acid sodium. The alkylsulfonic acid (or a salt thereof) typically is included in the aqueoussolution at a concentration of about 15 mM to about 300 mM, about 20 mMto about 200 mM, about 30 mM to about 150 mM, about 40 mM to about 120mM, about 50 mM to about 100 mM, and/or about 60 mM to about 90 mM.Other concentrations of the alkyl sulfonic acid (or salts thereof) mayalso be used. For example, when the sample is gradient eluted, onemobile phase may include the alkyl sulfonic acid (or salts thereof) at alower concentration and the second mobile phase may include the alkylsulfonic acid (or salts thereof) at a higher concentration. Thus, otherexemplary concentrations of the alkyl sulfonic acid (or salts thereof)in the aqueous solution include about 15 mM to about 50 mM, about 15 mMto about 40 mM, about 20 mM to about 30 mM, about 100 mM to about 300mM, about 120 mM to about 280 mM, and/or about 150 mM to about 250 mM.

A buffer typically is included in the aqueous solution at aconcentration of about 15 mM to about 200 mM, 20 mM to about 150 mM,and/or 30 mM to about 100 mM. Other concentrations of buffer may also beused. For example, when the sample is gradient eluted, one mobile phasemay include the buffer at a lower concentration and the second mobilephase may include the buffer at a higher concentration. Thus, otherexemplary concentrations of buffer in the aqueous solution include about15 mM to about 100 mM, about 20 mM to about 75 mM, about 25 mM to about50 mM, about 75 mM to about 125 mM, about 85 mM to about 115 mM, about100 mM to about 200 mM, and/or about 100 mM to about 150 mM. Exemplarybuffers include any buffer having a pK_(a) at an acidic pH, for example,a pH of about 1.5 to about 6, about 2 to about 5, about 2 to about 4,about 2 to about 3, and/or about 2 to about 2.5. Exemplary buffersinclude, but are not limited to, phosphate, citric acid/citrate, aceticacid/acetate, glycine, formic acid/formate, and succinic acid/succinate.In various embodiments, the buffer includes sodium phosphate.

Acetonitrile typically is included in the mobile phase in an amount ofabout 5% to about 95% by volume of the mobile phase, for example, about1% to about 30% by volume, about 2% to about 25% by volume, about 3% toabout 20% by volume, about 5% to about 15% by volume, about 8% to about12% by volume, and/or about 10% by volume. Other amounts of acetonitrilemay also be used. For example, when the sample is gradient eluted, onemobile phase may include acetonitrile at a lower concentration and thesecond mobile phase may include acetonitrile at a higher concentration.Thus, other exemplary amounts of acetonitrile include about 1% to about8% by volume, about 1% to about 6% by volume, about 1% to about 5% byvolume, about 2% to about 4% by volume, about 3% by volume, about 8% toabout 30% by volume, about 10% to about 25% by volume, about 15% toabout 20% by volume and/or about 18% by volume.

Methanol typically is included in the mobile phase in an amount of about5% to about 85% by volume of the mobile phase, for example, about 10% toabout 80% by volume, about 15% to about 75% by volume, about 20% toabout 70% by volume, about 25% to about 65% by volume, about 30% toabout 60% by volume, and/or about 35% to about 55% by volume. Otheramounts of methanol may also be used. For example, when the sample isgradient eluted, one mobile phase may include methanol at a lowerconcentration and the second mobile phase may include methanol at ahigher concentration. Thus, other exemplary amounts of methanol includeabout 5% to about 20% by volume, about 8% to about 15% by volume, about12% by volume, about 50% to about 85% by volume, about 60% to about 80%by volume, and/or about 72% by volume.

An exemplary mobile phase comprises about 5% to about 95% by volume ofan aqueous solution having a pH of about 2.0 to 3.0, the aqueoussolution comprising an alkyl sulfonic acid (e.g., 1-hexanesulfonic acidand/or 1-octanesulfonic acid) and a phosphate buffer, about 1% to about30% by volume of acetonitrile, and about 5% to about 85% by volume ofmethanol. In various embodiments, the aqueous solution includes about 15mM to about 300 mM of an alkyl sulfonic acid (e.g., 1-hexanesulfonicacid and/or 1-octanesulfonic acid), and about 15 mM to about 200 mMphosphate buffer.

The sample can be gradient eluted using first and second mobile phasesthat are mixed in different ratios throughout the elution. An exemplaryfirst mobile phase comprises about 75% to about 95% by volume of anaqueous solution having a pH of about 2.0 to 3.0, the aqueous solutioncomprising an alkyl sulfonic acid (e.g., 1-hexanesulfonic acid and/or1-octanesulfonic acid) and a phosphate buffer, about 1% to about 8% byvolume of acetonitrile, and about 5% to about 20% by volume of methanol.In various embodiments, the aqueous solution of the first mobile phaseincludes about 15 mM to about 50 mM of an alkyl sulfonic acid (e.g.,1-hexanesulfonic acid and/or 1-octanesulfonic acid), and about 15 mM toabout 100 mM phosphate buffer. An exemplary second mobile phasecomprises about 5% to about 30% by volume of an aqueous solution havinga pH of about 2.0 to 3.0, the aqueous solution comprising an alkylsulfonic acid (e.g., 1-hexanesulfonic acid and/or 1-octanesulfonic acid)and a phosphate buffer, about 8% to about 30% by volume of acetonitrile,and about 50% to about 85% by volume of methanol. In variousembodiments, the aqueous solution of the second mobile phase includesabout 100 mM to about 300 mM of an alkyl sulfonic acid (e.g.,1-hexanesulfonic acid and/or 1-octanesulfonic acid), and about 100 mM toabout 200 mM phosphate buffer.

In a gradient elution, the gradient typically starts with a highpercentage of the first mobile phase, for example, about 100% of thefirst mobile phase and 0% of the second mobile phase, about 90% of thefirst mobile phase and about 10% of the second mobile phase, and/orabout 80% of the first mobile phase and about 20% of the second mobilephase. Over a period of time, for example, over at least 5 minutes, overat least 10 minutes, over at least 15 minutes, over at least 20 minutes,over at least 25 minutes, and/or over at least 30 minutes, the ratio ofthe first mobile phase to the second mobile phase is continuouslydecreased. At the end of the period of time, the percentage of the firstmobile phase is lower than at the start of the gradient, for example,about 30% of the first mobile phase and about 70% of the second mobilephase, about 40% of the first mobile phase and about 60% of the secondmobile phase, about 50% of the first mobile phase and about 50% of thesecond mobile phase, about 60% of the first mobile phase and about 40%of the second mobile phase, and/or about 70% of the first mobile phaseand about 30% of the second mobile phase. In an exemplary elution at aflow rate of 1 mL/min, the elution profile provides 100% of the firstmobile phase for 2 minutes, then linearly decreases the amount of thefirst mobile phase to 60% and linearly increases the amount of thesecond mobile phase to 40% over a period of 18 minutes, then provides60% of the first mobile phase and 40% of the second mobile phase for 5minutes, and then provides 100% of the first mobile phase for 15minutes.

The sample is eluted from the column using a suitable flow rate, forexample, about 0.001 mL/min to about 2 mL/min, about 0.01 mL/min toabout 2 mL/min, about 0.1 mL/min to about 2 mL/min, about 0.5 mL/min toabout 2 mL/min, and/or about 1 mL/min. Further, the sample is eluted ata suitable column temperature, for example, about 20° C. to about 80°C., about 25° C. to about 60° C., about 30° C. to about 50° C., and/orabout 40° C.

The eluted sample is measured using a UV detector and the signal isrecorded using a suitable recording device. Typically, the eluted sampleis measured at a wavelength of about 170 nm to about 250 nm, about 170nm to about 240 nm, about 170 nm to about 230 nm, about 170 nm to about220 nm, about 170 nm to about 210 nm, 180 nm to about 250 nm, about 180nm to about 240 nm, about 180 nm to about 230 nm, about 180 nm to about220 nm, about 180 nm to about 210 nm, about 190 nm to about 250 nm,about 190 nm to about 240 nm, about 190 nm to about 230 nm, about 190 nmto about 220 nm, about 190 nm to about 210 nm, about 200 nm to about 250nm, about 200 nm to about 240 nm, about 200 nm to about 230 nm, about200 nm to about 220 nm, about 200 nm to about 210 nm, about 250 nm orless, about 240 nm or less, about 230 nm or less, about 220 nm or less,and/or about 210 nm or less.

Cysteamine Processing

The cysteamine formulation for analysis according to the inventivemethods includes all forms of cysteamine, including pharmaceuticallyacceptable salts. A “pharmaceutically acceptable salt” is a salt thatcan be formulated into a compound for pharmaceutical use, including butnot limited to metal salts (e.g., sodium, potassium, magnesium, calcium,etc.) and salts of ammonia or organic amines. Examples of cysteaminederivatives include hydrochloride, hydrobromide, acetate, maleate,pamoate, phosphate, methanesulfonate, p-toluenesulfonate, bitartrate andphosphocysteamine derivatives. An exemplary form of cysteamine iscysteamine bitartrate.

The cysteamine formulation can be an immediate release formulation.Immediate release formulations generally can be prepared for analysis bydissolving in a suitable solvent to form a sample solution. If needed,the immediate release cysteamine formulation can be ground or otherwisedeaggregated prior to dissolving. If the cysteamine formulation ispresent in a capsule or other individual dose unit, the capsule or otherunit can be opened and the contents emptied into a suitable vessel priorto dissolving.

The cysteamine formulation also can be a delayed release formulationsuch as an enteric-coated cysteamine formulation. To prepare delayedrelease cysteamine formulations such as enteric-coated cysteamine beads,the beads are generally ground to form a powder or are ground with anamount of a suitable solvent to form a paste. The resulting powder orpaste is then dissolved to form a sample solution for analysis.

Enteric coatings prolong release until the cysteamine product reachesthe intestinal tract, typically the small intestine. Because of theenteric coatings, delivery to the small intestine is improved therebyimproving uptake of the active ingredient while reducing gastric sideeffects. Exemplary enterically coated cysteamine products are describedin International Publication No. WO 2007/089670 published Aug. 9, 2007,which is incorporated in its entirety herein.

Generally, the enteric coating comprises a polymeric material thatprevents cysteamine product release in the low pH environment of thestomach but that ionizes at a slightly higher pH, typically a pH of 4 or5, and thus dissolves sufficiently in the small intestines to graduallyrelease the active agent therein. Accordingly, among the most effectiveenteric coating materials are polyacids having a pKa in the range ofabout 3 to 5. Suitable enteric coating materials include, but are notlimited to, polymerized gelatin, shellac, methacrylic acid copolymertype CNF, cellulose butyrate phthalate, cellulose hydrogen phthalate,cellulose proprionate phthalate, polyvinyl acetate phthalate (PVAP),cellulose acetate phthalate (CAP), cellulose acetate trimellitate (CAT),hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcelluloseacetate, dioxypropyl methylcellulose succinate, carboxymethylethylcellulose (CMEC), hydroxypropyl methylcellulose acetate succinate(HPMCAS), and acrylic acid polymers and copolymers, typically formedfrom methyl acrylate, ethyl acrylate, methyl methacrylate and/or ethylmethacrylate with copolymers of acrylic and methacrylic acid esters(Eudragit NE, Eudragit RL, Eudragit RS).

EXAMPLES Example 1

Cysteamine bitartrate samples were assessed by gradient elution HPLCusing an XBRIDGE C18 column (dimensions: 150 mm×4.6 mm; packing particlesize: 3.5 μm) (Waters, Milford, Mass.). The autosampler temperature was4° C. Approximately 10 μL or approximately 100 μL of sample was injectedonto the column. The column temperature was 40° C. and the sample waseluted at a flow rate of 1.0 mL/min according to the following profile:

HPLC Gradient Time (min) Mobile Phase A (%) Mobile Phase B (%) 0.0 100 02.0 100 0 20.0 60 40 25.0 60 40 25.1 100 0 40.0 100 0

Mobile Phase A contained 23.6 mM 1-octanesulfonic acid sodium and 29.0mM sodium phosphate (pH 2.6)/acetonitrile/methanol 85/3/12 (v/v/v).Mobile Phase B contained 0.20 M 1-octanesulfonic acid sodium and 0.10 Msodium phosphate (pH 2.6)/acetonitrile/methanol 10/18/72 (v/v/v). Thepurity of 1-octanesulfonic acid was >98%. Detection was carried outusing a UV detector at 210 nm.

Reference Solution Preparation.

Reference solutions of Cysteamine Bitartrate Analytical ReferenceStandard were prepared as follows. Working Standard and Working CheckStandard solutions were prepared having a nominal concentration of 0.54mg/mL Cysteamine Bitartrate Analytical Reference Standard in MobilePhase A using low actinic glassware. A Working Sensitivity solution wasprepared having a nominal concentration of 0.30 mg/mL CysteamineBitartrate Analytical Reference Standard in Mobile Phase A using lowactinic glassware, which corresponds to the limit of quantification(LOQ) for cysteamine. The water content of the Cysteamine BitartrateAnalytical Reference Standard was determined no more than 7 days beforeuse by Karl Fischer titration or thermal gravimetric analysis (TGA). TheReference Standard was stored refrigerated and blanketed under nitrogen.

Bead Prep Assay Sample Preparation.

Enteric-coated cysteamine (RP103) microbeads were prepared for analysisaccording to the following procedure. Enteric coated cysteamineformulation RP103 is described in co-owned application Ser. No. ______(Attny Docket No. 31075/47722) (herein incorporated by reference). About3.7 g of RP103 beads were ground to a fine powder using a ball mill forapproximately 1 minute at 27 Hz. The grind was transferred to an amberbottle for storage. Stock Bead Prep Assay sample solutions were preparedin duplicate by adding 370.4 mg±5 mg of the grind to a 250 mL lowactinic volumetric flask and diluting with Mobile Phase A. The mixturewas stirred with a stir bar for at least 15 minutes. Approximately 15 mLof the resulting solution was filtered through a 0.45 μm nylon filter,with the first 5 mL being discarded. The cysteamine concentration of theresulting Stock Bead Prep Assay sample solution was approximately 0.300mg/mL. Working Bead Prep sample solutions were prepared by placing 4.0mL of Stock Bead Prep Assay sample solution in a 25 mL low actinicvolumetric flask and diluting to volume with Mobile Phase A. Thecysteamine concentration of the resulting Working Bead Prep samplesolution was approximately 0.048 mg/mL.

Assay Sample Preparation.

Capsules containing enteric-coated cysteamine (RP103) microbeads wereprepared for analysis according to the following procedure. To reduceexposure to light and oxygen, sample preparation (from the initialweighing of the full capsules to the loading of sample vials on theHPLC) was completed in one day. Ten capsules were weighed. The capsulecontents were emptied and the empty shells were weighed to determine theaverage capsule fill weight. The capsule contents were ground to a finepowder using a ball mill for approximately 1 minute at 27 Hz. The grindwas transferred to an amber bottle for storage. Stock sample solutionswere prepared in duplicate by adding the appropriate amount of the grindfor 1 capsule (as determined by the average capsule fill weight) to a 25mL low actinic volumetric flask and diluting with Mobile Phase A. Themixture was stirred with a stir bar for at least 15 minutes. Theresulting solution was centrifuged at about 3400 rpm for 5 minutes.Approximately 15 mL of the centrifuged solution was filtered through a0.45 μm nylon filter (Acrodisc, 25 mm diameter), with the first 5 mLbeing discarded, to obtain Stock sample solutions. Working samplesolutions were prepared by placing 6.0 mL of Stock sample solution (for25 mg capsules) or 2.0 mL of Stock sample solution (for 75 mg capsules)in a 10 mL low actinic volumetric flask and diluting to volume withMobile Phase A.

Content Uniformity Sample Preparation.

Capsules containing enteric-coated cysteamine (RP103) microbeads wereprepared for analysis according to the following procedure. To reduceexposure to light and oxygen, sample preparation (from the initialweighing of the full capsules to the loading of sample vials on theHPLC) was completed in one day. Ten capsules were weighed. The contentsof each capsule were emptied into separate mortars and the empty shellswere weighed to determine the individual capsule fill weight. About 1-2mL of Mobile Phase A was added into the mortar. The beads wereimmediately ground to a paste. If needed, additional Mobile Phase A wasadded to the paste, up to 5 mL total. The paste was transferred to a 250mL low actinic volumetric flask. The mortar and pestle were thoroughlyrinsed with Mobile Phase A and the rinse solution was collected in tothe same flask. The flask was filled about three-quarters full withMobile Phase A and stirred for at least 15 minutes. The flask was filledto volume with Mobile Phase A. Approximately 20 mL of the resultingsolution was filtered through a 0.45 μm nylon filter (Acrodisc, 25 mmdiameter), with the first 5 mL being discarded, to obtain Stock CUsample solutions. Working CU sample solutions were prepared by placing12.0 mL of Stock CU sample solution (for 25 mg capsules) or 4.0 mL ofStock CU sample solution (for 75 mg capsules) in a 25 mL low actinicvolumetric flask and diluting to volume with Mobile Phase A. Thecysteamine concentration of the resulting Working CU sample solutionswas approximately 0.048 mg/mL.

Data Analysis.

The cysteamine Working Standard solution concentration was calculatedaccording to the following equation: Cysteamine Concentration(C_(std))=mg Cysteamine Bitartrate Analytical ReferenceStandard×P_(f)/25.0 mL

P_(f) represents a purity factor for the standard material. P_(f) wascalculated according to the following equation:

P _(f) =B×(100−Water)×C/100

where B=the anhydrous cysteamine free base in the Cysteamine BitartrateAnalytical Reference Standard (expressed as a decimal value on thestandard bottle label),water=the water content as determined by Karl Fischer or TGA no morethan 7 days before use (expressed as a percentage), andC=the cystamine correction (expressed as a decimal value on the standardbottle label).

The amount of cysteamine per capsule was calculated according to thefollowing equation:

mg cysteamine per capsule=(A _(Sam) /A _(Std))×C _(Std)×DF×(AveWt/SamWt)

where A_(sam)=the peak area of cysteamine in the sample chromatogramwith a 10 μL injection,A_(std)=the average peak area of cysteamine in all Working Standardsolution chromatograms with a 10 μL injection,C_(Std)=the concentration (mg/mL) of cysteamine in the Working Standardsolution,DF=the dilution factor (125 for 75 mg capsules; 41.6667 for 25 mgcapsules),AveWt=the average capsule fill weight (mg), andSamWt=the sample weight (mg).

For Content Uniformity, the amount of cysteamine per capsule wascalculated according to the following equation:

mg cysteamine per capsule=(A _(Sam) /A _(Std))×C _(std)×DF

where A_(Sam)=the peak area of cysteamine in the sample chromatogramwith a 10 μL injection,A_(Std)=the average peak area of cysteamine in all Working Standardsolution chromatograms with a 10 μL injection,C_(std)=the concentration (mg/mL) of cysteamine in the Working Standardsolution, andDF=the dilution factor (1562.5 for 75 mg capsules; 520.8 for 25 mgcapsules).

For the Bead Prep Assay, the amount of cysteamine per capsule wascalculated according to the following equation:

mg cysteamine per capsule=(A _(Sam) /A _(Std))×C _(std)×DF×(AveWt/SamWt)

where A_(Sam)=the peak area of cysteamine in the sample chromatogramwith a 10 μL injection,A_(Std)=the average peak area of cysteamine in all Working Standardsolution chromatograms with a 10 μL injection,C_(Std)=the concentration (mg/mL) of cysteamine in the Working Standardsolution,DF=the dilution factor (use the 75 mg Dilution Factor, 1562.5),AveWt=the average capsule fill weight (mg) (use the target fill weight,370.4 mg), andSamWt=the sample weight (mg) (use the actual weight used in samplepreparation).

The percentage of the label claim (% LC) was calculated for the Assay,Content Uniformity, and Bead Prep Assay sample solutions according tothe following equation:

% LC=(mg cysteamine)/LC×100%

where mg cysteamine=the amount calculated by the applicable equationabove, andLC=the amount of the label claim 75 mg or 25 mg) (use 75 mg for the BeadPrep Assay).

The amount of substances related to cysteamine bitartrate (includingcysteamine impurities) such as cystamine was calculated according to thefollowing equation:

mg related substance=(A _(RS) /A _(Std))×(C _(Std)/RRF)×DF×(AveWt/SamWt)

where A_(RS)=the peak area of any related substance in the Workingsample solution chromatogram with a 100 μL injection (peaks before RRT0.48 were disregarded; peaks observed in the chromatogram of the secondinjection of Mobile Phase A/Blank (100 μL injection) were alsodisregarded),A_(Std)=the average peak area of cysteamine in all Working Standardsolution chromatograms with a 10 μL injection,C_(Std)=the concentration (mg/mL) of cysteamine in the Working Standardsolution,RRF=the relative response factor (0.98 for cystamine; 1.00 for otherrelated substances),DF=the dilution factor (12.5 for 75 mg capsules; 4.16667 for 25 mgcapsules),AveWt=the average capsule fill weight (mg), andSamWt=the weight the sample grind from the Working sample solutionpreparation (mg).

The weight percentage of cystamine and other individual relatedsubstances was determined according to the following equation:

% individual related substance=mg related substance/mg cysteamine×100%

where mg related substance=the amount of related substance calculatedabove, andmg cysteamine=the amount of cysteamine for the Assay sample.

The percentage of total related substances was determined by summing allrelated substances greater than or equal to 0.05%. Peaks after 28minutes were disregarded. In contrast to a previous electrochemicaldetection method that disregarded early-eluting peaks as not relevant tothe purity calculation, the foregoing method determines that early peaksare impurities and integrates early-eluting peaks as described above.

Five lots of enteric-coated cysteamine (RP103) (delayed release, 75 mgstrength) and two lots of cysteamine without an enteric-coating(Cystagon®) (immediate release) were tested according to the proceduredescribed above to determine the weight percentage of relatedsubstances. The five RP103 batches were tested at the time of lotrelease (“Time 0”) and at 12 or 18 months after lot release. Thepercentage of each related substance was calculated as a weightpercentage of cysteamine. An RRF of 1.00 was assigned to each relatedsubstance (except for cystamine) because a sufficient quantity of therelated substances to determine RRF was not isolated. For cystamine, RRFwas determined to be 0.98. The results of testing are provided in Table1 and a representative chromatogram is provided in FIG. 1.

TABLE 1 Relative Cystagon ® Cystagon ® Related Retention RP103 Lot 1RP103 Lot 2 RP103 Lot 3 RP103 Lot 4 RP103 Lot 5 Lot 1 Lot 2 SubstanceTime Time Time Time Time 12 Time 12 Not Not Peak No. (RRT) 0 18 mo. 0 18mo. 0 18 mo. 0 mo. 0 mo. available available Cystamine n/a 3.2 4.2 3.44.1 3.2 3.8 3.7 3.7 3.6 3.3 9.1 5.2 A 0.48-0.52 0.14 0.13 0.07 B0.55-0.56 <0.05 0.21 0.19 0.06 0.08 0.09 0.10 C 0.67-0.69 0.08 0.07 30.78-0.82 0.17 0.13 0.18 0.16 0.14 0.14 0.06 0.10 0.07 0.11 0.09 10.83-0.86 0.35 0.71 0.30 0.49 0.31 0.46 0.21 0.45 0.20 0.50 0.31 0.20 D0.87-0.90 0.13 2 0.91-0.94 0.09 0.94 0.09 1.1 0.09 1.1 0.06 0.71 0.060.75 0.15 4 0.95-0.97 0.09 0.22, 0.22, 0.08 0.07 0.06 0.06 5 1.13-1.160.14 0.17 0.16 0.10 0.08 0.07 E 1.25-1.26 0.11 6 1.30-1.34 0.19 0.210.21 0.09 0.07 0.14 0.07 F 1.51-1.54 0.05 0.07 0.06 0.07 G 1.59-1.630.07 0.06 0.11 0.11 0.09 0.07 H 1.64-1.65 0.09 0.12 0.11 I^(§) 1.72-1.731.0^(§) 1.0^(§) 0.07 J 1.98-2.01 0.12 K 2.39-2.41 0.08 Individual 0.93,0.10% 1.56, 1.77, 1.39, Un- 1.39, 0.06% 0.05% 0.05% 0.06% specified1.93, 0.06% Peak 2.16, 0.07% 2.20, 0.05% Total 4.0 7.1 4.2 6.8 3.9 6.44.2 5.4 4.2 5.0 10.3 5.7 Related (with- (with- Substances out out PeakI) Peak I) ^(§)Peak I is believed to be a contaminant and was notobserved in samples analyzed on other days.

These results demonstrate lower total related substances forenteric-coated cysteamine at the time of lot release and at 12 monthsafter lot release relative to cysteamine without an enteric-coating. Forexample, the present method shows that lots of an immediate releasecysteamine formulation comprise over 5% cystamine impurity whereas itwas previously shown by an electrochemical detection method that theimmediate release product comprised less than 5% cystamine impurity. Incontrast, the method shows that the delayed release composition RP103comprises less than 5% cystamine impurity. Further, using the UVdetection method additional impurity peaks, for example, Peak 1, Peak 5,and Peak 6 (see Table 1 and FIG. 1), were detectable, which were notdetected by an electrochemical detection method. These results alsodemonstrate that the present methods provides highly sensitive andaccurate determination of related substances present in the cysteamineformulations.

Example 2

UV detection of cysteamine bitartrate impurities was compared toelectrochemical detection, which has been used previously to detectimpurities in an immediate release formulation of cysteamine. Twocysteamine bitartrate samples (without an enteric coating) were assessedby HPLC. The first sample (Sample A) contained cysteamine bitartrate(CBT) at a concentration of 1 mg/mL, spiked with thiomorpholine (TMP),thiomorpholine-1-oxide (OTMP), Peak 1 (see Table 1 and FIG. 1)(RRT0.85), and Peak 4 (see Table 1 and FIG. 1) (R4). The second sample(Sample B) contained cysteamine bitartrate (CBT) at a concentration of 1mg/mL, spiked with lanthionamine (TBEA), Peak 5 (see Table 1 and FIG. 1)(R5), Peak 6 (see Table 1 and FIG. 1) (R6), and Peak D (see Table 1)(RRT0.89). Both samples were stored at 5° C. for a few days prior toHPLC analysis.

Samples A and B were assessed by isocratic elution HPLC using anATLANTIS T3 C18 column (dimensions: 150 mm×4.6 mm; packing particlesize: 3 μm) (Waters, Milford, Mass.). The autosampler temperature was 5°C. Approximately 10 μL of sample was injected onto the column. Thecolumn temperature was 40° C. and the sample was eluted at a flow rateof 1.0 mL/min for 15 minutes. The mobile phase contained 8.8 mM sodiumhexanesulfonate monohydrate, 0.1% H₃PO₄ in 88/12 water/acetonitrile(v/v). The needle was washed with water.

UV detection was carried out using a UV detector at 200 nm.Electrochemical detection was carried out using an electrochemicaldetector with a condition cell (250 mV, 5 μA) and an analytical cell(650 mV, 100 μA).

Electrochemical detection was initially thought to be a more sensitivedetection method compared to UV detection. However, as shown in FIG. 2and FIG. 3, additional peaks were detected and greater peak resolutionwas demonstrated by the UV detection method described herein (A) ascompared to the electrochemical detection method (B). FIG. 2 correspondsto Sample A and FIG. 3 corresponds to Sample B.

Without intending to be bound by theory, it is believed that many of theimpurities identified herein and detected using the UV detector at lowwavelength do not undergo a redox reaction, which is necessary for animpurity to be detected using an electrochemical detector. Therefore, asillustrated below, the UV detection method identifies more impuritypeaks than an electrochemical detection method.

For example, the UV detection method identified additional impuritypeaks not identified by electrochemical detection, such asthiomorpholine (TMP), thiomorpholine-1-oxide (OTMP), Peak D, Peak 4,lanthionamine (TBEA), Peak 5, and Peak 6.

Thus, the UV detection method described herein demonstrated highersensitivity and less selectivity compared to electrochemical detection.

What is claimed is:
 1. A method of analyzing purity of a compositioncomprising cysteamine comprising: (i) injecting a sample solutioncomprising cysteamine onto a reverse-phase HPLC column; (ii) eluting thesample from the column using a mobile phase comprising an alkyl sulfonicacid, a buffer, acetonitrile, and methanol; and (iii) measuring theeluted sample using a UV detector at a wavelength of about 170 nm toabout 250 nm.
 2. The method of claim 1, further comprising dissolving acysteamine sample in a solvent having an acidic pH to form the samplesolution comprising cysteamine.
 3. A method of analyzing purity ofenteric-coated cysteamine beads comprising: (i) grinding enteric-coatedcysteamine beads; (ii) dissolving the ground beads in a solvent havingan acidic pH to form a sample solution comprising cysteamine; (iii)injecting the sample solution onto a reverse-phase HPLC column; (iv)eluting the sample from the column using a mobile phase comprising analkyl sulfonic acid, a buffer, acetonitrile, and methanol; and (v)measuring the eluted sample using a UV detector at a wavelength of about170 nm to about 250 nm.
 4. The method of claim 2 or 3, wherein thesolvent comprises an alkyl sulfonic acid, a buffer, acetonitrile, andmethanol.
 5. The method of any of the preceding claims, wherein the HPLCcolumn is selected from the group consisting of a C18 column, a C8column, a silica column, a cyano-bonded silica column, and aphenyl-bonded silica column.
 6. The method of any of the precedingclaims, wherein the HPLC column has a packing material particle size ofabout 1 μm to about 10 μm in diameter.
 7. The method of any of thepreceding claims, wherein the HPLC column has a packing materialparticle size of about 2 μm to about 5 μm in diameter.
 8. The method ofany of the preceding claims, wherein the HPLC column has an internaldiameter of about 0.1 mm to about 10 mm.
 9. The method of any of thepreceding claims, wherein the HPLC column has an internal diameter ofabout 1 mm to about 5 mm.
 10. The method of any of the preceding claims,wherein the HPLC column has a length of about 5 mm to about 500 mm. 11.The method of any of the preceding claims, wherein the HPLC column has alength of about 50 mm to about 250 mm.
 12. The method of any of thepreceding claims, wherein the eluted sample is measured using a UVdetector at a wavelength of about 180 nm to about 230 nm.
 13. The methodof any of the preceding claims, wherein the eluted sample is measuredusing a UV detector at a wavelength of about 190 nm to about 210 nm. 14.The method of any of the preceding claims, wherein the mobile phasecomprises: about 5% to about 95% by volume of an aqueous solution havinga pH of about 2.0 to 3.0, the aqueous solution comprising an alkylsulfonic acid and a phosphate buffer, about 1% to about 30% by volume ofacetonitrile, and about 5% to about 85% by volume of methanol.
 15. Themethod of claim 14, wherein the aqueous solution comprises: about 15 mMto about 300 mM of an alkyl sulfonic acid, and about 15 mM to about 200mM phosphate buffer.
 16. The method of any of the preceding claims,wherein the sample is eluted using a gradient elution.
 17. The method ofclaim 16, comprising gradient eluting the sample using first and secondmobile phases, wherein the first mobile phase comprises: about 75% toabout 95% by volume of an aqueous solution having a pH of about 2.0 to3.0, the aqueous solution comprising an alkyl sulfonic acid and aphosphate buffer, about 1% to about 8% by volume of acetonitrile, andabout 5% to about 20% by volume of methanol; and the second mobile phasecomprises: about 5% to about 30% by volume of an aqueous solution havinga pH of about 2.0 to 3.0, the aqueous solution comprising an alkylsulfonic acid and a phosphate buffer, about 8% to about 30% by volume ofacetonitrile, and about 50% to about 85% by volume of methanol.
 18. Themethod of claim 17, wherein the aqueous solution of the first mobilephase comprises: about 15 mM to about 50 mM of an alkyl sulfonic acid,and about 15 mM to about 100 mM phosphate buffer.
 19. The method ofclaim 17, wherein the aqueous solution of the second mobile phasecomprises: about 100 mM to about 300 mM of an alkyl sulfonic acid, andabout 100 mM to about 200 mM phosphate buffer.
 20. The method of any ofthe preceding claims, wherein the sample is eluted using a flow rate ofabout 0.001 mL/min to about 2 mL/min.
 21. The method of any of thepreceding claims, wherein the sample is eluted at a column temperatureof about 20° C. to about 80° C.
 22. The method of any of the precedingclaims, wherein the sample is eluted at a column temperature of about30° C. to about 50° C.
 23. The method of any of the preceding claims,wherein the sample solution comprises cysteamine bitartrate.
 24. Themethod of claim 3, wherein the beads are ground to form a powder. 25.The method of claim 3, wherein the beads are ground to form a paste. 26.The method of any of the preceding claims, wherein the alkyl sulfonicacid is selected from the group consisting of ethanesulfonic acid,propanesulfonic acid, butanesulfonic acid, pentanesulfonic acid,hexanesulfonic acid, heptanesulfonic acid, octanesulfonic acid,nonanesulfonic acid, and decanesulfonic acid.
 27. The method of any ofthe preceding claims, wherein the alkyl sulfonic acid is selected fromthe group consisting of 1-hexanesulfonic acid and 1-octanesulfonic acid.28. A method of analyzing purity of compositions comprising cysteaminecomprising: (i) dissolving a cysteamine sample in a solvent having anacidic pH to form a sample solution comprising cysteamine; (ii)injecting the sample solution onto a C18 reverse-phase HPLC column;(iii) gradient eluting the sample from the column using first and secondmobile phases, wherein the first mobile phase comprises: about 85% byvolume of an aqueous solution having a pH of about 2.6, the aqueoussolution comprising about 23.6 mM 1-octanesulfonic acid sodium and about29 mM sodium phosphate; about 3% by volume of acetonitrile; and about12% by volume of methanol, and the second mobile phase comprises: about10% by volume of an aqueous solution having a pH of about 2.6, theaqueous solution comprising about 0.2 M 1-octanesulfonic acid sodium andabout 0.1 M sodium phosphate; about 18% by volume of acetonitrile; andabout 72% by volume of methanol; and (iv) measuring the eluted sampleusing a UV detector at a wavelength of about 210 nm or less.
 29. Themethod of claim 28, comprising gradient eluting at a flow rate of about1.0 mL/min according to the following profile: HPLC Gradient Time (min)First Mobile Phase (%) Second Mobile Phase (%) 0.0 100 0 2.0 100 0 20.060 40 25.0 60 40 25.1 100 0 40.0 100 0


30. The method of claim 28 or 29, wherein the HPLC column has a packingmaterial particle size of about 3.5 μm in diameter.
 31. The method ofany one of claims 28 to 30, wherein the HPLC column has a length ofabout 150 mm and an internal diameter of about 4.6 mm.
 32. The method ofany of the preceding claims, comprising injecting at least 30 μg ofcysteamine onto the column.